One form of common chromosome mutation is aneuploidy wherein the number of individual chromosomes present in a cell either increases or decreases from that present in a normal cell. The absence of one chromosome from the diploid complement is called "monosomy". The presence of an extra chromosome is called "trisomy". Trisomy 21 is a condition wherein there exists an extra chromosome 21. This trisomy is the most common form of aneuploidy and gives rise to Down's syndrome which is the congenital manifestation of severe mental retardation.
Generally, the diagnosis of Down's syndrome and other aneuploidies requires obtaining fetal cells by amniocentesis or chorionic villus sampling. This requires routine cytogenetic procedures which include the necessity of cell culture (up to 7-14 days), chromosome preparation and karyotyping. This process is lengthy, expensive and labor intensive. Recently, a molecular cytogenetic technique for detecting aneuploidy, namely, fluorescent in situ hybridization (FISH) has been developed. Although FISH is relatively fast and accurate, performance of the FISH method requires highly trained technicians and expensive equipment and reagents. In U.S. Pat. No. 5,213,961 by Bunn et al., is disclosed a method of quantitative PCR by competitive methodology. In that invention, the parameters affecting DNA amplification and a mechanism to distinguish differences in template (both test and control) ratios and copy numbers are discussed. The Bunn disclosure addressed as a primary object of the invention such parameters and their effect on the amplification process. These parameters were believed to arise predominantly from the nature of the DNA primers and their respective primer binding sites. The invention disclosed that it is necessary for such primers and binding sites of the control and test DNA sequences to be functional equivalents of one another. Emphasis was placed on the fact that PCR amplification was initiated utilizing identical primers for the control and test sequences. Moreover, that invention discussed the capacity to distinguish the test sequence from the control sequence by changing the size of the control sequence (by either deletion or insertion) as much as 100 to 200 base pairs. The Bunn invention also discussed altering the control sequence such as by substitution of sequence by site specific mutagenesis either creating or destroying an restriction enzyme cleavage site. It was reasoned that if the primers and binding sites were functional equivalents, then the amplification process could progress equivalently at similar rates (even if the control sequence was longer or shorter than the test sequence).
The invention disclosed in the Bunn patent further assumed that the described method would not be dependent upon variables which normally affect PCR amplification and that such method would therefore allow quantitation between template species (i.e. control and test sequence) regardless of such usual variables as long as the reaction would give good amplification of template DNA. However, the Bunn invention overlooked critical elements in the relative amplification rates that are inherently introduced by altering control and test sequence primer site lengths, control template sequence lengths, and guanine (G) and cytosine (C) content of such sequences. Moreover, the detection methodology of that invention disclosed and discussed only such means as are compatible with identification of DNA species via ethydium bromide, radioactivity, or colorimetric technology in conjunction with gel electrophoresis techniques and the like. Thus, the reasoning contemplated for introducing point mutations in the control sequence was based solely on a desire to create restriction enzyme sites as a means by which amplified DNA segments could be distinguishable from one another based solely on size.
In another invention, PCT application WO 94/03638 by Mansfield, a method is disclosed whereby aneuploidy may be detected by utilization of short tandem repeat DNA sequences present in chromosome DNA. In that invention, PCR methodology was utilized to amplify the short tandem repeat sequences. There are, however, two limitations for this method. One drawback is that not all Down's patients are heterozygous at the polymorphic site. About 25% of Down's patients are caused by a meiosis II nondisjunction error wherein two of the three chromosome 21s present in the cells are genetically identical. Therefore, the homozygosity resulting from this error will render a significant portion of the affected patient population unidentifiable by the proposed method. The second limitation of the disclosed method is that it differentiates alleles based on the size of the polymorphic PCR products. The problem here, as described above, is that the detection method must be capable of distinguishing size differences and also that because smaller fragments amplify more readily, errors can arise when calculating ratios. In some instances, larger species may be over shadowed by the amplification of the smaller species. This is possible even where the same PCR primers are used to amplify DNA sequence from the same or from different alleles.
In yet another example, determination of gene-dosage by PCR was disclosed (Genomics 21, 304-310, 1994 Francesco, C. et al.) wherein internal control DNA sequence was designed as a deletion mutant of the wildtype sequence. Again, quantitative analysis was dependant upon gel electrophoresis and measuring radioactivity of the different sized products.
Each of the above examples fail to consider the significant effect of amplification rate differences that even a small change in molecule size or G and C content in DNA bases have on the ultimate quantity of DNA segments resulting from a plurality of PCR thermocycles. Moreover, each of the above methodologies requires detection of the amplified species by first electrophoresing the amplified DNA to separate the amplified species. Thus, there is still a need in the art of quantitative PCR as such technology relates to the detection of aneuploidy, and other chromosomal anomalies, for a methodology which can accurately determine gene copy number without the occurrence of unreliable results derived from factors that inherently affect amplification of which template length, G and C content and ultimate detection methodology are primary components.